以納豆菌發酵生產凝乳酵素之研究 張宸瑋、施英隆
E-mail: 321997@mail.dyu.edu.tw
摘 要
近幾年來，由於犢牛凝乳?〞瘧Y重不足，細菌所生產的凝乳?﹞w經廣泛的被接受成為犢牛凝乳?〞煽壎N品之一。本論文 研究是藉由納豆菌 ( Bacillus subtilis natto Takahashi ) 的固態及液態培養，來獲得環境因子對其生產凝乳?〞獐v響，並以回應 曲面法 ( RSM )探討生產因子之最佳化，同時以超過濾濃縮裝置、電泳 ( SDS-PAGE ) 進行部份純化及分子量分析，並進行 酵素活性影響因子之影響。 由納豆菌的固態培養可以得知，在 37℃、pH 6、溼度 70% 的環境下，以米殼和基本鹽類來培 養 72 小時之後，有最高的凝乳比率。而由納豆菌的液態培養可以得知，在37℃、pH 6、轉速 175 rpm 的環境下，以澱粉
、玉米抽出物、黃豆粉、乾奶粉以及基本鹽類來培養 72 小時之後，有最高的凝乳比率。 由回應曲面法的結果可以得知，
在配方組成濃度分別為：澱粉 55.41 g/L、玉米抽出物 1.5 g/L、黃豆粉 2.69 g/L 以及乾奶粉 22.29 g/L 時，有最高的凝乳 活性 1048.02 SU/mL。經由超過濾濃縮裝置以及電泳分析之後，可以得知納豆菌所生產的凝乳?﹞壑l量介於 20000 – 30000 g/mole 之間。 納豆菌所生產的凝乳?’b牛奶基質溫度為 60℃、pH 6 時，有最佳的酵素活性；在 60℃ 加熱 60 分鐘 或者 70℃ 加熱 5分鐘之後，此凝乳?◎|喪失 80% 以上的酵素活性。而由 pH 對其酵素活性的影響結果中可以得知，在 pH 5 – pH 6 之間，此酵素的凝乳活性則是相當的穩定。 由各金屬離子對納豆菌凝乳?〞獄簿嶼〝宎v響結果中可以得知，一 價陽離子對其凝乳活性並無顯著影響，但是二價陽離子卻是有明顯的促進凝乳活性的效果。而在添加汞離子時，此酵素會 馬上失去其酵素活性，所以汞離子為此納豆菌凝乳?〞漣磻蹌砥C
關鍵詞 : 凝乳?﹛B納豆菌、回應曲面法、凝乳比率、酵素活性、抑制劑 目錄
封面內頁 簽名頁 授權書iii 中文摘要iv 英文摘要vi 誌謝viii 目錄ix 圖目錄xiv 表目錄xvii 第一章 緒論1 1.1 研究背景1 1.1.1 納 豆菌1 1.1.2 凝乳?? 1.2 研究目標3 1.3 研究架構4 1.3.1 培養基之篩選4 1.3.2 MCE ( Milk-clotting enzyme ) 發酵因子探討5 1.3.3 以RSM ( Response surface methodology ) 對 MCE 之探討6 1.3.4 MCE 的濃縮、電泳分析以及酵素活性因子分析7 第二章 文 獻回顧8 2.1 酵素性凝乳8 2.1.1 酪蛋白8 2.1.2 酵素性凝乳之凝固機制10 2.1.3 影響凝乳物性與凝析現象之因子12 2.2 凝乳??9 2.2.1 凝乳?◇媄?9 2.2.2 凝乳?﹞屁A用性23 2.2.3 凝乳?﹞妍虓~化24 2.3 回應曲面法27 2.3.1 設計原理28 2.3.2 二水準因子設 計( two-level factorial design )28 2.3.3 陡升路徑法( Method of path of Steepest Ascent )30 2.3.4 中心混成設計( Central Composite Design )30 2.3.5 數據統計分析( Regression model analysis )31 第三章 研究材料與方法33 3.1 實驗材料及儀器設備33 3.1.1 實驗 藥品33 3.1.2 實驗儀器35 3.1.3 實驗器材36 3.2 實驗培養方法38 3.2.1 菌株來源38 3.2.2 菌株活化38 3.2.3 固態特殊培養39 3.2.4 液態特殊培養40 3.3 實驗分析方法44 3.3.1 酵素活性分析44 3.3.1.1 凝乳活性44 3.3.1.2 蛋白分解活性45 3.3.2 酵素活性 影響因子分析48 3.3.2.1 最適作用溫度48 3.3.2.2 最適作用 pH 值48 3.3.2.3 熱安定性48 3.3.2.4 pH 安定性49 3.3.2.5 各金屬離 子之影響49 3.3.3 超過濾濃縮裝置系統50 3.3.4 回應曲面法51 3.3.4.1 2 因子設計( two-level factorial design )51 3.3.4.2 陡升實驗 設計( Method of path of Steepest Ascent )53 3.3.4.3 中心混成實驗設計( Central Composite Design )55 3.3.4.4 二次回歸分析(
Regression model analysis )57 第四章 結果與討論58 4.1 培養基篩選58 4.1.1 固態培養基58 4.1.2 液態培養基60 4.1.3 培養基篩 選的結果討論62 4.2 MCE發酵因子探討63 4.2.1 培養時間63 4.2.1.1 固態培養基63 4.2.1.2 液態培養基65 4.2.1.3 培養時間的結 果討論69 4.2.2 培養溫度70 4.2.2.1 固態培養基70 4.2.2.2 液態培養基72 4.2.2.3 培養溫度的結果討論75 4.2.3 培養 pH 值77 4.2.3.1 液態培養基77 4.2.3.2 培養 pH 值的結果討論80 4.2.4 培養轉速值82 4.2.4.1 液態培養基82 4.2.4.2 培養轉速值的結果討 論85 4.3 RSM 實驗設計86 4.3.1 2 因子設計結果86 4.3.2 陡升設計結果90 4.3.3 中心混成實驗設計結果92 4.3.4 中心混成實驗 二階回歸分析結果討論94 4.4 MCE 的濃縮、電泳分析以及酵素活性影響因子分析105 4.4.1 超過濾濃縮裝置105 4.4.2 電泳 ( SDS-PAGE ) 實驗分析結果討論110 4.4.3 酵素活性影響因子113 4.4.3.1 最適作用溫度113 4.4.3.2 最適作用 pH 值116 4.4.3.3 熱安定性118 4.4.3.4 pH 安定性122 4.4.3.5 各金屬離子之影響126 第五章 結論128 5.1 培養基的篩選128 5.2 環境因子的影 響128 5.3 培養基最佳組成比例129 5.4 超過濾濃縮129 5.5 電泳分析130 5.6 酵素活性影響因子130 參考文獻132
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